This invention relates to the field of preservation of biological materials and, more particularly, to compositions and methods for the preservation of living organs, tissues and cells from mammals, marine organisms and plants.
Methods for the preservation of biological materials are employed in many clinical and veterinary applications wherein living material, including organs, tissues and cells, is harvested and stored in vitro for some period of time before use. Examples of such applications include organ storage and transplants, autologous and allogeneic bone marrow transplants, whole blood transplants, platelet transplants, embryo transfer, artificial insemination, in vitro fertilization, skin grafting and storage of tissue biopsies for diagnostic purposes. Preservation techniques are also important in the storage of cell lines for experimental use in hospital, industrial, university and other research laboratories.
Methods currently employed for the preservation of cellular biological materials include immersion in saline-based media; storage at temperatures slightly above freezing; storage at temperatures of about xe2x88x9280xc2x0 C.; and storage in liquid nitrogen at temperatures of about xe2x88x92196xc2x0 C. The goal of all these techniques is to store living biological materials for an extended period of time with minimal loss of normal biological structure and function.
Storage of organs, such as heart and kidneys, at temperatures below 0xc2x0 C. frequently results in the loss of many cells with a corresponding reduction in viability of the organ. Such complex biological materials are therefore typically stored in aqueous, saline-based media at temperatures above freezing, typically around 4xc2x0 C. Saline-based media typically consist of isotonic saline (sodium chloride 0.154 M) which has been modified by the addition of low concentrations of various inorganic ions, such as sodium, potassium, calcium, magnesium, chloride, phosphate and bicarbonate, to mimic the extracellular environment. Small amounts of compounds such as glucose, lactose, amino acids and vitamins are often added as metabolites. All saline-based media used for preservation of biological materials have high electrical conductivity. Examples of media currently employed for the preservation of biological materials include phosphate-buffered saline (PBS), M-2 (a Hepes buffered murine culture medium), Ringer""s solution and Krebs bicarbonate-buffered medium.
The viability of biological materials stored in saline-based media gradually decreases over time. Loss of viability is believed to be due to the build-up of toxic wastes, and loss of metabolites and other supporting compounds caused by continued metabolic activity. Using conventional saline-based media, living tissues can only be successfully preserved for relatively short periods of time. Examination of the microstructure of organs stored towards the upper limit of time shows degeneration, such as of mitochondria in heart muscle, and the performance of the organ once replaced is measurably compromised. For example, a human heart can only be stored in cold ionic solutions for about 5 hours following removal from a donor, thereby severely limiting the distance over which the heart can be transported.
When employing freezing techniques to preserve biological materials, high concentrations (approximately 10% by volume) of cryoprotectants, such as glycerol, dimethylsulfoxide (DMSO), glycols or propanediol, are often introduced to the material prior to freezing in order to limit the amount of damage caused to cells by the formation of ice crystals during freezing. The choice and concentration of cryoprotectant, time-course for the addition of cryoprotectant and temperature at which the cryoprotectant is introduced all play an important role in the success of the preservation procedure. Furthermore, in order to reduce the loss of cells, it is critical that such variables as the rate and time-course of freezing, rate and time-course of thawing and further warming to room or body temperature, and replacement of cryoprotectant solution in the tissue mass with a physiological saline solution be carefully controlled. The large number of handling steps required in freezing techniques increases the loss of cells. The freezing techniques currently employed in the preservation of biological materials are both technically demanding and time consuming. Other disadvantages of preserving biological materials by freezing include: reduction of cell viability; potential toxic effects of the cryoprotectant to the patient upon re-infusion; and the high costs of processing and storage.
As an example, cryopreservation, generally including the addition of DMSO as a cryoprotectant, is presently used to store bone marrow harvested for use in transplantation procedures following, for example, high dose chemotherapy or radiotherapy. In autologous transplants the bone marrow must be preserved for prolonged periods, ranging from weeks to months. However, this technique results in significant reduction of stem cell recovery, to levels as low as 50% or less. An additional disadvantage of this technique is that significant damage to various mature cells can occur, thereby requiring further processing to remove these cells prior to freezing. Finally, the use of DMSO results in moderate to severe toxicity to the patient on re-infusion of the preserved bone marrow.
There thus remains a need in the art for improved methods for the preservation of living biological materials.
The present invention provides compositions and methods for preserving living biological materials that enable materials including organs, tissues and cells to be stored for extended periods of time with minimal loss of biological activity.
In one aspect, the present invention provides solutions for preserving the viability of living biological materials, comprising a first neutral solute with no net charge, having a molecular weight of at least about 335 and a solubility in water of at least about 0.3 M; and a second neutral solute having a molecular weight of less than about 200, the second solute additionally having both hydrophilic and hydrophobic moieties.
In a preferred embodiment, the first neutral solute is either a disaccharide or a trisaccharide, preferably selected from the group consisting of raffinose, trehalose, sucrose, lactose and analogs thereof. The analogs may be either naturally occurring or synthetic. The second neutral solute is preferably selected from the group consisting of trimethyl amino oxide (TMAO), betaine, taurine, sarcosine, glucose, mannose, fructose, ribose, galactose, sorbitol, mannitol, inositol and analogs thereof. Most preferably, the first neutral solute is selected from the group consisting of raffinose and trehalose, and the second neutral solute is selected from the group consisting of trimethyl amine oxide (TMAO) and betaine. While it is not an endogenous osmolyte of cells and is not taken up by them, polyethylene glycol molecular weight 1500, (hereinafter referred to as PEG 1500) may be substituted for TMAO or betaine in all the preservative solutions of the present invention.
Preservation solutions of the present invention may also include one or more ions. In one embodiment, the preservation solutions employed in the inventive methods also comprise sodium sulfate and calcium, the calcium preferably being present as calcium sulfate or calcium chloride at a concentration of more than about 1.5 mM or less than about 2.0 mM. Preferably the calcium chloride is present at a concentration of about 1.5 mM to about 2.0 mM, most preferably about 1.75 mM.
While the preferred solution for the preservation of a biological material will depend upon the specific biological material to be preserved, in one aspect it has been found that solutions comprising either, raffinose and TMAO, raffinose and betaine, or trehalose and TMAO are particularly efficacious in the preservation of many biological materials. In one embodiment, the inventive solutions comprise raffinose and either TMAO, betaine or PEG 1500 in an osmolar ratio of less than about 2.0:1 or more than about 1.1:1. Preferably the preservative solutions of this aspect comprise raffinose and either TMAO or betaine in an osmolar ratio between about 1.1:1 to about 2.0:1, more preferably about 1.4:1 to about 1.8:1, and most preferably about 1.6:1. Preferably, the solutions of this aspect of the present invention comprise TMAO or betaine at a concentration of about 70-75 mM, most preferably about 72 mM; raffinose at a concentration of about 120-130 mM, most preferably about 126 mM; sodium sulphate at a concentration of about 35-45 mM, most preferably about 39 mM; and calcium sulphate at a concentration of about 1.5-2.0 mM, most preferably about 1.75 mM.
In another embodiment, the inventive preservation solutions comprise a first neutral solute and a second neutral solute as defined above, preferably raffinose and TMAO, in combination with an equiosmolar amount of sodium citrate, replacing sodium sulphate, and with calcium chloride, the calcium chloride preferably being present at a concentration of more than about 1.5 mM or less than 2.0 mM, more preferably at a concentration from about 1.5 mM to about 2.0 mM, and most preferably about 1.75 mM. Preferably, the solution comprises more than about 25 mM and less than about 35 mM sodium citrate, more preferably between about 25 mM and about 35 mM sodium citrate.
In another aspect, the present invention provides solutions for preserving the viability of living biological materials, comprising either TMAO or PEG 1500, in combination with sodium chloride and calcium chloride. In one embodiment, the preservation solutions comprise TMAO at a concentration of more than about 150 mM or less than about 230 mM, more preferably at a concentration of between about 150 mM and about 230 mM and most preferably at a concentration of between about 160 mM and about 215 mM; sodium chloride at a concentration of more than about 30 mM or less than about 60 mM, more preferably between about 30 mM and about 60 mM and most preferably at a concentration of about 46.8 mM; and calcium chloride at a concentration of more than about 1.5 mM or less than about 2.0 mM, more preferably at a concentration between about 1.5 mM and about 2.0 mM, and most preferably at a concentration of about 1.75 mM.
In a further aspect, the present invention provides solutions for the preservation of living biological materials that comprise either betaine, trimethyl amine oxide (TMAO) or PEG 1500 as the principal organic component and sodium chloride as the principal inorganic component. In certain embodiments, such solutions comprise either, betaine, TMAO or PEG 1500 and sodium chloride, together with sodium citrate and/or a calcium salt. In a preferred embodiment, such solutions comprise betaine or TMAO, sodium chloride and sodium citrate, with the betaine or TMAO preferably being present at a concentration greater than about 150 mM or less than 220 mM, more preferably between about 150 mM and about 220 mM, and most preferably at a concentration of about 184 mM for TMAO or 187 mM for betaine; the sodium citrate preferably being present at a concentration greater than about 1.5 mM or less than about 2.5 mM, more preferably between about 1.5 mM and about 2.5 mM, and most preferably at a concentration of about 1.96 mM; and the sodium chloride preferably being present at a concentration greater than about 35 mM or less than about 55 mM, more preferably between about 35 mM and about 55 mM, and most preferably at a concentration of about 45.8 mM. Such solutions have been found to be particularly efficacious in the preservation of platelets.
The present invention further provides methods for lyophilizing living biological materials that enable the materials to be stored in an inactive, desiccated state at room temperature for extended periods of time with minimal loss of biological activity. In certain embodiments, such methods are employed to preserve eukaryotic cells, including eukaryotic cells that are encapsulated in a cell wall, such as plant cells. The methods comprise contacting, preferably immersing, the biological material to be preserved in one or more of the preservative solutions of the present invention. The solution containing the biological material is then rapidly cooled to a temperature of less than about xe2x88x9280xc2x0 C., more preferably less than about xe2x88x92140xc2x0 C., and most preferably to a temperature of about xe2x88x92196xc2x0 C., and dried to provide a freeze-dried material. The cooled material is preferably dried by sublimation under a high vacuum to provide a freeze-dried material having less than about 5%, more preferably less than about 1%, by weight of residual water. In one embodiment of the present invention, the biological material is cooled rapidly following immersion in the preservative solution, most preferably by plunging into liquid nitrogen, and is dried under conditions which minimize increases in temperature before the removal of water is complete.
In yet another aspect, a method for the treatment of leukemia is provided, the method comprising removing bone marrow from a patient, contacting the bone marrow with a preservation composition or solution of the present invention for a period of at least about 3 days at a temperature of less than about 0xc2x0 C., more preferably between about xe2x88x924xc2x0 C. and about xe2x88x9280xc2x0 C., and most preferably at a temperature of about xe2x88x9280xc2x0 C., in order to purge the bone marrow of leukemic cells, and returning the purged bone marrow to the patient.
As detailed below, it has been found that the solutions and methods of the present invention can be employed to maintain the viability of living biological materials, including cells, tissues and organs, for longer periods of time than are generally possible with conventional preservation methods, thereby providing improved storage and transport times for biological materials for use in applications such as organ transplants and bone marrow transplants.
The preservation methods of the present invention are less complex than many of the methods typically employed for the preservation of biological materials, thereby reducing costs and increasing the ease of use and availability of preservation procedures. Furthermore, the inventive compositions are of low toxicity, resulting in fewer negative side effects when biological materials, such as transplant organs, are returned to a patient.
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description, read in conjunction with the accompanying drawings.